Urany-Less Low Voltage Transmission Electron Microscopy: A Powerful Tool for Ultrastructural Studying of Cyanobacterial Cells.

Sample preparation protocols for conventional high voltage transmission electron microscopy (TEM) heavily rely on the usage of staining agents containing various heavy metals, most commonly uranyl acetate and lead citrate. However high toxicity, rising legal regulations, and problematic waste disposal of uranyl acetate have increased calls for the reduction or even complete replacement of this staining agent. One of the strategies for uranyless imaging is the employment of low-voltage transmission electron microscopy. To investigate the influence of different imaging and staining strategies on the final image of cyanobacterial cells, samples stained by uranyl acetate with lead citrate, as well as unstained samples, were observed using TEM and accelerating voltages of 200 kV or 25 kV. Moreover, to examine the possibilities of reducing chromatic aberration, which often causes issues when imaging using electrons of lower energies, samples were also imaged using a scanning transmission electron microscopy at 15 kV accelerating voltages. The results of this study demonstrate that low-voltage electron microscopy offers great potential for uranyless electron microscopy.

Comparative assessment of environmental impacts of 1st generation (corn feedstock) and 3rd generation (carbon dioxide feedstock) PHA production pathways using life cycle assessment.

Polyhydroxyalkanoates (PHA) are bio-based and biodegradable alternatives to conventional plastic types and have the potential to reduce the environmental impacts along the life cycle. In comparison to already established production routes for PHA (heterotrophic production) based on renewable feedstock like glucose (first generation feedstock), novel production routes, such as the photoautotrophic production of PHA based on CO2 as feedstock (third generation feedstock) could offer new perspectives with regard to the reduction in the environmental impacts. To quantify the environmental impacts of PHA produced via photoautotrophic and heterotrophic production pathways, life cycle assessment (LCA) methodology based on ISO 14040/44 was applied, thus conducting a first of its kind comparative study for PHA based on third generation feedstock. The results show that the photoautotrophic production of PHA has advantages in comparison to heterotrophic PHA based on glucose originating from corn as feedstock in all the assessed environmental impact categories, thus showing the environmental potential of novel production routes for bioplastics. Additionally, the results of the LCA show that the chloroform-based extraction method, commonly used in the downstream processes of both the technologies, has a significant contribution of environmental impacts in the production of PHA. Therefore, the reduction of chloroform loss during the extraction process can reduce its environmental impact. Our results indicate that PHA production from CO2 using the photoautotrophic production route is a promising technology with regard to the environmental impacts when compared to the heterotrophic production based on glucose feedstock.

PHB Producing Cyanobacteria Found in the Neighborhood-Their Isolation, Purification and Performance Testing.

Cyanobacteria are a large group of prokaryotic microalgae that are able to grow photo-autotrophically by utilizing sunlight and by assimilating carbon dioxide to build new biomass. One of the most interesting among many cyanobacteria cell components is the storage biopolymer polyhydroxybutyrate (PHB), a member of the group of polyhydroxyalkanoates (PHA). Cyanobacteria occur in almost all habitats, ranging from freshwater to saltwater, freely drifting or adhered to solid surfaces or growing in the porewater of soil, they appear in meltwater of glaciers as well as in hot springs and can handle even high salinities and nutrient imbalances. The broad range of habitat conditions makes them interesting for biotechnological production in facilities located in such climate zones with the expectation of using the best adapted organisms in low-tech bioreactors instead of using "universal" strains, which require high technical effort to adapt the production conditions to the organism's need. These were the prerequisites for why and how we searched for locally adapted cyanobacteria in different habitats. Our manuscript provides insight to the sites we sampled, how we isolated and enriched, identified (morphology, 16S rDNA), tested (growth, PHB accumulation) and purified (physical and biochemical purification methods) promising PHB-producing cyanobacteria that can be used as robust production strains. Finally, we provide a guideline about how we managed to find potential production strains and prepared others for basic metabolism studies.

In situ based surface-enhanced Raman spectroscopy (SERS) for the fast and reproducible identification of PHB producers in cyanobacterial cultures.

The production of polyhydroxybutyrate (PHB) by autotrophic fermentation of cyanobacteria has received increasing interest in the light of carbon emission reducing process strategies. Biotechnological approaches are in development to optimize the yield of PHB, including adapted cultivation media, characterized by a limitation of key nutrients: cyanobacteria accumulate PHB as energy storage molecules under limited growth conditions. Since there is an increasing demand for fast, simple and reliable analytics, we report the establishment of surface enhanced Raman spectroscopy (SERS) as a suitable monitoring tool for up scaled PHB production processes. Both, pure Ag-colloids mixed with bacterial culture, and in situ prepared colloids (Ag-Synechocystis), generated on the cell surface directly, were successfully applied and evaluated for this purpose. SERS measurements with in situ prepared Ag-colloids improved the reproducibility of Raman signals from 54.8% to 93.9%. The measurement time could be reduced significantly, completing our secondary goal. The quality of classically and in situ prepared Ag-colloids was monitored by zeta potential measurements and scanning electron microscopy (SEM) respectively. For data interpretation and statistical model-building an in house written code in the open source software RStudio was implemented. It was applied for the differentiation of PHB producers at the cellular level, revealing heterogeneities within sample groups regarding the PHB amount accumulated. The results obtained using the statistical model were validated as well and were complementary to the reference HPLC analysis. Therefore, a fast and reliable identification in situ SERS tool for the selection of the most promising cyanobacterial PHB production was established.

Novel unexpected functions of PHA granules.

Polyhydroxyalkanoates (PHA), polyesters accumulated by numerous prokaryotes in the form of intracellular granules, have been for decades considered being predominantly storage molecules. However, numerous recent discoveries revealed and emphasized their complex biological role for microbial cells. Most of all, it was repeatedly reported and confirmed that the presence of PHA granules in prokaryotic cells enhances stress resistance and robustness of microbes against various environmental stress factors such as high or low temperature, freezing, oxidative, and osmotic pressure. It seems that protective mechanisms of PHA granules are associated with their extraordinary architecture and biophysical properties as well as with the complex and deeply interconnected nature of PHA metabolism. Therefore, this review aims at describing novel and unexpected properties of PHA granules with respect to their contribution to stress tolerance of various prokaryotes including common mesophilic heterotrophic bacteria, but also extremophiles or photo-autotrophic cyanobacteria. KEY POINTS: • PHA granules present in bacterial cells reveal unique properties and functions. • PHA enhances stress robustness of bacterial cells.

Characterization of polyhydroxyalkanoates produced by Synechocystis salina from digestate supernatant.

The current commercial production of polyhydroxyalkanoates (PHA) is based on heterotrophic bacteria, using organic carbon sources from crops. To avoid the competition with food and feed production, cyanobacteria, metabolising PHA from carbon dioxide can be used. This research focuses on the investigation of the thermal and rheological properties of PHA polymers accumulated by Synechocystis salina, which had been cultivated in digestate supernatant and a mineral medium. The dried bacterial cells had a polymer content of 5.5-6.6%. The relevance of the derived PHA polymers for the common melt polymer processing was correlated with their molecular mass distribution as well as with their thermal and rheological properties. The determined thermal and rheological properties showed that PHA polymers accumulated by S. salina on digestate supernatant or mineral medium are comparable with the commercial available poly(3-hydroxybutyrate). However, the results showed that PHA polymers in general require modification before melt processing to increase their stability in the molten state.

Results from a round robin test for the ecotoxicological evaluation of construction products using two leaching tests and an aquatic test battery.

A European round robin test according to ISO 5725-2 was conceptually prepared, realised, and evaluated. The aim was to determine the inter-laboratory variability of the overall process for the ecotoxicological characterization of construction products in eluates and bioassays. To this end, two construction products BAM-G1 (granulate) and HSR-2 (roof sealing sheet), both made of EPDM polymers (rubber), were selected. The granular construction product was eluted in a one stage batch test, the planar product in the Dynamic Surface Leaching test (DSLT). A total of 17 laboratories from 5 countries participated in the round robin test: Germany (12), Austria (2), Belgium (1), Czech Republic (1) and France (1). A test battery of four standardised ecotoxicity tests with algae, daphnia, luminescent bacteria and zebrafish eggs was used. As toxicity measures, EC50 and LID values were calculated. All tests, except the fish egg test, were basically able to demonstrate toxic effects and the level of toxicity. The reproducibility of test results depended on the test specimens and the test organisms. Generally, the variability of the EC50 or LID values increased with the overall level of toxicity. For the very toxic BAM-G1 eluate a relative high variability of CV = 73%-110% was observed for EC50 in all biotests, while for the less toxic HSR-2 eluate the reproducibility of EC50 varied with sensitivity: it was very good (CV = 9.3%) for the daphnia test with the lowest sensitivity, followed by the algae test (CV = 36.4%). The luminescent bacteria test, being the most sensitive bioassay for HSR-2 Eluate, showed the highest variability (CV = 74.8%). When considering the complex overall process the reproducibility of bioassays with eluates from construction products was acceptable.

Formation and ecotoxicity of N-heterocyclic compounds on ammoxidation of mono- and polysaccharides.

Ammoxidation of technical lignins under mild conditions is a suitable approach to artificial humic substances. However, carbohydrates as common minor constituents of technical lignins have been demonstrated to be a potential source of N-heterocyclic ecotoxic compounds. Ethyl acetate extracts of ammoxidation mixtures of the monosaccharides glucose and xylose exhibited considerable growth inhibiting activity in the OECD 201 test, with 4-methyl-1H-imidazole, 4-(hydroxymethyl)-1H-imidazole, and 3-hydroxypyridine being the most active compounds. The amount of N-heterocyclic compounds formed at moderate ammoxidation conditions (70 °C, 0.2 MPa O2, 3 h) was significantly lower for the polysaccharides cellulose and xylan (16-30 µg/g of educt) compared to glucose (15.4 mg). Ammoxidation at higher temperature is not recommendable for carbohydrate-rich materials as much higher amounts of N-heterocyclic compounds were formed from both monosaccharides (100 °C: 122.4-160.5 mg/g of educt) and polysaccharides (140 °C: 5.52-16.03 mg/g of educt).